Purification and antibiotic inhibition of L,d transpeptidase in Enterobacter cloacae | Poster Board #2009

β-lactam antibiotics such as penicillin have been particularly important in modern medicine through their ability to disrupt cross-linkage activity within bacterial cell walls. They work by binding to penicillin-binding proteins, or PBPs, which prevents cross-linkage between peptide strands of the cell wall from occurring, resulting in an overall weaker cell wall which subsequently kills bacterial cells. Enterobacter cloacae also expresses several L,D transpeptidases or Ldts, which can also cross-link peptidoglycan through an alternative pathway and are not as easily recognized by β-lactams. This project aims to quantify the activity of LdtEC in the presence of different β-lactam antibiotics, specifically carbapenems. LdtEC was over-expressed and purified as a recombinant protein from an E. coli expression system. Nitrocefin was used to quantify enzyme activity and inhibition in the presence of various β-lactam antibiotics. Based on that data, we also propose to perform assays to determine the minimum-inhibitory concentrations of antibiotic necessary to inhibit the activity of LdtEC. Through quantification of these interactions, it was determined that meropenem and nafcillin had the greatest inhibitory effect on LdtEC. While a related carbapenem, imipenem, showed no inhibition, suggesting that the substituents on the carbapenem core play a critical role in enabling Ldt inhibition.